| Abstract
| - Thrombopoietin (TPO) is a hematopoietic factor involved in theregulation of megakaryocytopoiesis. Full length recombinant human TPO (332 residues) hasbeen expressed in BHK cells andpurified to homogeneity using conventional means. Peptide,disulfide, and glycosylation mapping ofhuman TPO from residues 1 to 246 has been carried out using liquidchromatography−electrospray massspectrometry (LC−ESMS). A modification of the ramped orificemethod of Carr and co-workers [Carret al. (1993) Protein Sci. 2, 183−196] is employed,providing additional information for assignment ofthe LC−ESMS chromatograms. With the modification, b- andy-series peptide ions are produced viafront-end CID which confirms the mass-based assignments. Theresults of our analysis of TPO indicatethat the amino acid sequence of TPO 1−246 is as expected from thetransfected cDNA with completecleavage of the signal peptide. Two unique disulfides are formedbetween the four cysteines in the cytokinedomain of TPO: Cys7−Cys151 and Cys29−Cys85. Theglycosylation map indicates the position,occupancy, and structures of the N- and O-glycansin TPO 1−246. In addition, site specific structuralcharacterization of the PNGase F-liberated N-glycans hasbeen performed following purification by high-pH anionic exchange chromatography with pulsed amperometric detection(HPAEC-PAD); the resultscorroborate the LC−ESMS data. The N-glycans are ofthe complex type with the core-fucosylateddisialylated biantennary and trisialylated triantennary structurespredominating. The O-glycans are ofthe mucin type with the monosialylated and disialylated GalGalNAc-S/Tstructures predominating.Furthermore, we propose that the C-terminal domain of TPO befurther divided into two domains on thebasis of sequence homology among the cloned sequences andglycosylation/structural features: anN-glycandomain (154−246) and an O-glycan domain(247−332).
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